Expression of GABAAα1, GABAB1, and mGluR2 receptors in the lateral geniculate body of male neonates born to diabetic rats

Objective(s): Diabetes during gestation is one of the most common pregnancy complications and has adverse effects on offspring, including a negative impact on the offspring’s central nervous system (CNS). Diabetes is a metabolic disease associated with visual impairment. Due to the importance of the lateral geniculate body (LGB) in the visual pathway, the present study examined the effect of maternal diabetes on the expression of gamma-aminobutyric acid (GABAAα1 and GABAB1) and metabotropic Glutamate (mGlu2) receptors in the LGB of male neonates of diabetic rats. Materials and Methods: Diabetes was induced in female adult rats by a single intraperitoneal dose of streptozotocin (STZ) 65 (mg/kg). In the Insulin-treated diabetic rats, diabetes was controlled by subcutaneous NPH-insulin injection daily. After mating and delivery, male offspring were killed by carbon dioxide gas inhalation at P0, P7, and P14 (postnatal days 0, 7, and 14). The expression of GABAAα1, GABAB1, and mGluR2 in the LGB of male neonates was determined using the immunohistochemistry (IHC) method. Results: The expression of GABAAα1 and GABAB1 was significantly reduced, whereas the expression of mGluR2 was markedly increased in the diabetic group compared with the control and insulin-treated groups at P0, P7, and P14. Conclusion: The results of the present study showed that induction of diabetes altered the expression of GABAAα1, GABAB1, and mGluR2 in the LGB of male neonates born to diabetic rats at P0, P7, and P14. Moreover, insulin treatment could reverse these effects of diabetes.


Introduction
Diabetes during pregnancy is associated with an increased risk of neurological disorders in offspring (1). Among the complications of diabetes, visual disturbances, and vision loss are of particular importance (2). The subcortical visual system consists of a large group of nuclei including the lateral geniculate body (LGB) (3).
LGB is a structure located in the metathalamus and is responsible for the connection between the optic nerve and the primary visual cortex. LGB nuclei are divided into a dorsal and a ventral part. Both of these subsets receive signals from retinal ganglion cells that are transmitted by the optic nerve, however, only the dorsal part transmits the signals to the visual neocortex (4,5).
LGB contains different neurotransmitters (6) and its function depends on tight control of these excitatory/inhibitory neurotransmitter levels. The impaired balance of excitatory/ inhibitory neurotransmitters in the neural network plays an important role in the pathophysiology of a wide variety of neurodevelopmental disorders (7). Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) (8,9). GABA receptors are present throughout the visual pathway (10). Two structural types of GABA receptors including GABA A and GABA C are ionotropic but the GABA B receptors are metabotropic. GABA A consists of 19 subunits that are encoded by different genes, including α, β, δ, ϒ, ε, and σ. Subunits α 1 and ϒ 2 have the highest expression among all subunits. The GABA B receptors include GABA B1a , GABA B1b , and GABA B2 . (11,12). GABA receptors are present in LGB at the early postnatal stages. The high-affinity uptake of GABA displayed a noticeable peak on a postnatal day 15 in rat LGB (6). GABA receptor subunits including GABA Aα1 and GABA B , and Glutamic acid decarboxylase (GAD) expression significantly decreased in diabetic rats' cerebral cortex (13). Glutamate is the main excitatory neurotransmitter in the mammalian CNS including LGB (6,14). Glutamate receptors are divided into two groups, including ionotropic and metabotropic receptors (15). There are eight types of metabotropic receptors (mGluR 1 -mGluR 8 ) that belong to the G proteincoupled receptor superfamily (GPCRs), which are classified into three groups (16). Glutamate expression increases in the visual cortex and LGB after birth and reaches the activity level of this receptor in adults 15 to 20 days after birth (6).
Group II mGluRs are found in the thalamus (and LGB as a thalamic nucleus) and are main to circuit behavior in this structure, group II mGluRs modulate the thalamic relay to the cortex. Postsynaptic mGluR subtypes exhibit different distributions and may have clear functional roles in the visual system. Thalamocortical neurons in the LGB dynamically transmit visual signals from the retina to the neocortex, and this function can be modulated by the activation of mGluRs. glutamate receptors (mGluRs), which can be activated by retinogeniculate and corticothalamic pathways in the LGB (17). Liu Z. et al. (2019) reported that diabetes is associated with abnormalities in glutamate receptors in the brain that cause neuronal degeneration (18). Diabetes can affect the central visual system, including LGB, in rats (19). The effects of diabetes on the central visual system are due to high blood glucose. High blood glucose of a diabetic mother easily enters the fetus through the placenta during pregnancy, so it is reasonable to imagine that it can affect the developing nervous system of the fetus too. Since the effects of maternal diabetes on the expression of neurotransmitter receptors involving the visual system of neonates have not been studied yet, we decided to investigate the distribution pattern of GABA Aα1 , GABA B1, and mGluR2 receptors using the IHC technique in the LGB of male rat neonates born to diabetic mothers.

Materials and Methods
All experiments involving animals were accomplished by the guidelines of the National Institutes of Health (NIH) and accepted by the Committee for the Use of Laboratory Animals in Mashhad University of Medical Sciences (MUMS), Mashhad, Iran (IR.MUMS.medical. REC.1398.794).

Animals
Female Wistar rats (body weight: 200-250 g; age range: 6-8 weeks) were obtained from the animal house of Mashhad University of Medical Sciences (Mashhad, Iran). Animals were maintained in a room at 23±2 °C (55±5% humidity, with a 12 hr light/12 hr darkness cycle) with standard water and feed. Two female rats in the same group were kept in a cage. The animals (n=15) were randomly divided into three groups: 1. Control (Con; n=5), 2. Diabetic (Dia; n=5), 3. Insulin-treated diabetic (Ins; n=5).

Diabetes induction
Diabetes was induced by an intraperitoneal injection of a single dose (65 mg /kg) of streptozotocin (STZ, Sigma Aldrich, Hamburg, Germany) diluted in normal saline in both the Dia and the Ins groups (20). The fasting blood glucose was measured by tail blood sampling 72 hr after STZ injection by a commercial digital glucometer (Accu-chek®, Germany). The rats with blood glucose levels of more than 150 mg/dl were considered diabetic and were divided into two groups, including Dia and Ins (21). In the Dia group, the blood glucose level was measured by collecting blood from the end of the caudal vein daily, the mean blood glucose level in the Dia group was 358.3±84.24 mg/dl, in addition, signs of binge drinking were observed in the rats of this group. The rats in the Ins group received 2-4 units of protamine-zinc insulin (NPH) (Exir Pharmaceutical Company, Iran) subcutaneously twice a day after becoming diabetic (22). Injectable insulin doses were determined by measuring daily blood glucose to make sure that the rats' blood glucose levels were kept within the normal range. The mean blood glucose level in the Ins group was 105.3±10.14 mg/dl. To eliminate the potential effect of injection, the Con group rats received intraperitoneal injections of normal saline. Female rats in all three groups were caged with healthy male rats (body weight: 250-300 g; age range: 16 weeks), one week after developing diabetes in the Dia group and after diabetes control in the Ins group. Animals were allowed to give birth naturally at the end of the pregnancy period; the birthday was considered P0 (23). Rat pups born to diabetic and insulin-treated diabetic mothers were fed by healthy mothers to eliminate the possible effects of milk from diabetic rats and thus focus only on the fetal period environment (24). Male neonates in each group were randomly divided into three subgroups: P0 (n=5), P7 (n=5), and P14 (n=5).

Tissue preparation
Male neonates at the P0, P7, and P14 subgroups were sacrificed using CO 2 inhalation on postnatal days 0, 7, and 14, respectively. The animal brains were removed from the skull and fixed in a 10% formalin solution for 72 hr. After fixation, the brains were dehydrated using ascending concentration ethanol (70 to 100%) and then immersed in paraffin. Coronal brain sections including LGB were prepared with a thickness of five micrometers.

Immunohistochemistry
ScyTek-ACV999 kit was used to perform immunohistochemistry (for details see below). The LGB sections were deparaffinized using xylene. The sections were hydrated using descending ethanols (100 to 70%), then washed with 0.1 M phosphate-buffered saline (PBS). Antigen retrieval was performed using Tris Buffered Saline 1% (TBS 1X, T5912-Sigma) at 96 °C for 15 min, and then washed with PBS three times.
To inhibit endogenous peroxidase, the sections were treated with 3% hydrogen peroxide (Sigma-7722-84-1) in methanol for 10 min, and then the slides were washed three times with PBS. Subsequently, the sections were incubated overnight at 4 °C with the primary antibody diluted in PBS. Primary antibodies included: GABABR1 antibody: orb85203, GABA A Receptor alpha 1 antibody: orb10677, MGLUR2 antibody: orb85332.
Then, slides were washed with PBS and incubated with 100 µl from Linker (Diagnostic BioSystems-PVP1000D) for 15 min. In the next stage, the sections of LGB were incubated with DAB (3, 3′-Diaminobenzidine) solution (ScyTek-ACV999), then washed with water. The sections were counterstained with Harris hematoxylin for 10 sec, then were again washed with water, dehydrated using ascending ethanol, clarified with Xylene, and finally, covered with coverslips (25,26).

Quantitative analysis
Tissue sections were examined by means of an optical microscope (Olympus BX51, Japan) linked to a camera (Olympus DP12, Japan) by ND25 and OP filters.
LGB boundaries in rat neonates were delineated by crossreferencing with the classical atlases of the rat brain (26-28) (Figure 1). Quantitative analysis of the immunohistochemical staining intensity in LGB was evaluated by ImageJ software. Using the subsequent formula calculated optical density (OD) for further statistical analysis.

Diabetes affects expression of LGB receptors
Alipour et al.

Optical Density = log (Max intensity/Mean intensity)
The maximum intensity in Red-Green-Blue images is 255 and the mean intensity was measured by ImageJ software (18,29).

Statistical analysis
Data were analyzed using GraphPad Prism 9 software, and they were compared through one-and two-way ANOVA, as well as post hoc Tukey's test. All data were expressed as mean±SEM. A P<0.05 was considered statistically significant.

GABA B1 expression in LGB
In the LGB, the main effect on the expression of GABA B1 in treatment groups was significantly different [F (2, 36)=22.21, P<0.0001]. The expression of GABA B1 was significantly decreased at P7 and P14 in the Dia group compared with the Con group (P<0.01 and P<0.0001, respectively). In the Ins group, the expression of GABA B1 significantly increased compared with the Dia group at P7 and P14 (P<0.05 and P<0.0001, respectively). No remarkable difference was perceived in the expression of this receptor in the LGB between rat neonates of the Ins and the Con groups at P0, P7, and P14. The main effect was significantly different in postnatal days [F (2, 36)=209.0, P<0.0001]. In all three groups, the OD of GABA B1 receptor expression in the LGB significantly increased from P0 to P7 and from P7 to P14 (each, P<0.0001). A significant difference was not seen in the main interaction effect of treatment groups×postnatal days [F (4, 36)=2.593, P=0.0527]. The highest OD was for the Con group at P14 (0.5310±0.02045), and the lowest OD was for the Dia group at P0 (0.5119±0.01421) (Figure 2).

GABA Aα1 expression in the LGB
The main effect on the expression of GABA Aα1 in treatment groups showed a significant difference [F (2, 36)=46.89, P<0.0001]. In the Dia group, the OD of the GABA Aα1 expression was remarkably decreased at P0, P7, and P14 compared with the Con group (P<0.05 for P0 and P<0.0001 for P7 and P14). The expression of GABA Aα1 increased in LGB of rat neonates in the Ins group compared with the Dia group at P7 and P14 (each, P<0.0001). A remarkable difference was not detected in the expression of GABA Aα1 between rat neonates of the Ins and Con groups at P0, P7, and P14. In all three groups, the OD of GABA Aα1 receptor expression in the LGB significantly increased at P7 compared with P0 (P<0.0001). The main effect was    (Figure 3).

Expression of mGluR2 in the LGB
The results of the present study demonstrated that the main effect on the expression of mGluR2 in treatment groups was significantly different [F (2, 36)=35.17, P<0.0001]. The expression of mGluR2 in the Dia group remarkably increased compared with the Con group at P7 and P14 (each, P<0.0001). In the Ins group, expression of mGluR2 significantly decreased compared with the Dia group at P7 and P14 (P<0.0001 and P<0.001, respectively). No remarkable difference was detected in the expression of this receptor at P0, P7, and P14 between rat neonates of the Ins and the Con groups. The main effect was significantly different on postnatal days [F (2, 36)=862.3, P<0.0001]. In all three groups, the OD of mGluR2 receptor expression in the LGB significantly increased from P0 to P7 and from P7 to P14 (each, P<0.0001). No significant difference was found in the main interaction effect of treatment groups× postnatal days [F (4, 36)=4.018, P=0.0085]. The highest OD expression was for the Dia group at P14 (0.4445±0.05368) and the lowest OD expression was for the Con group at P0 (0.4131±0.04770) (Figure 4).

Discussion
Diabetes in pregnancy has detrimental consequences on the development of the CNS in children (1). Visual impairment and vision loss are among the consequences of diabetes in individuals (30). Diabetes usually leads to neural impairment and also leads to changes in the expression of neurotransmitters (13), including GABA and glutamate content (31), which have an essential role in visual functions in LGB. In the visual system, GABA-containing inhibitory interneurons in the LGN innervate thalamocortical relay neurons by axodendritic and dendrodendritic synapses, also mGluR-mediated actions influence thalamic relay neurons (32). In diabetes, changes in GABA content and glutamate may be related to visual dysfunctions (31). Since no research has been found on the alteration of GABAergic   LGB) which has been displayed by the brown color is different on different days at P0, P7, and P14 in different groups (Con: neonates of control rats, Dia: neonates of diabetic rats, Ins: neonates of diabetic rats treated with insulin) The expression of mGluR2 was significantly increased at P7 and P14 in the Dia group compared to the Con group. No remarkable difference was perceived in the expression of this receptor in the LGB between rat neonates of the Ins and the Con groups at P0, P7, and P14. Immunoreaction visualization with DAB, counterstaining with Harris hematoxylin; scale bars=100 μm (A). Immunohistochemically staining intensity of mGluR2 was expressed as Optical Density. Values represent the mean±SD. The level of significance between groups showed by # (### and #### mean P<0.001 and P<0.0001, respectively). The level of significance between postnatal days showed by * (* means P<0.0001) (B)

Diabetes affects expression of LGB receptors
Alipour et al.
and glutamatergic receptors expression in the visual system of neonates of diabetic mothers, in this study we examined GABA Aα1, GABA B1, and mGluR2 in the LGB of neonates born to diabetic mothers.

Effects of maternal diabetes on the expression of GABA receptors in the LGB of neonates
The development of the central nervous system depends on a number of crucial functions performed by GABAergic signaling. There is evidence to suggest that the variety of GABAergic signaling expands during postnatal development to provide the variety and accuracy of inhibitory processes needed for the growing demands of the maturing brain (33). The lateral geniculate nucleus is responsible for transmitting visual information from the retina to the visual cortex. Several modulatory inputs, including GABAergic inputs, affect how this information is processed. Local interneurons within the LGB and extrathalamic projections from the thalamic reticular nucleus are both sources of GABAergic modulation. Through the activation of GABA A and GABA B receptors, these inputs can modify the characteristics of the receptive field and regulate visual attention. GABA receptor heterogeneity's significance for brain function is still not fully understood (25). In a study on patients with schizophrenia, Jong H. Yoon et al. found that schizophrenia is accompanied by impaired visual inhibition. They discovered lower GABA concentrations in schizophrenia patients' visual cortex, demonstrating both a deficit in GABA concentrations and a correlation between GABA levels and a behavioral measure of visual inhibition (34). Also, it is possible that in our study, GABA reduction in the LGB following diabetes also reduces visual-related symptoms. Since no studies similar to our work were found, a relationship between diabetes and the expression of GABA receptors in brain regions including the hippocampus has been demonstrated. A study (2019) reported that diabetes reduced GABA receptors expression in the hippocampus of rats. GABA receptor treatment improves the effects of blood glucose on weight gain, blood glucose, and the structure of neurons in the hippocampus (35). Biju et al. (1998) also reported that in the brain regions of adult rats inhibitory neurotransmitters (GABA) decreased (36). Also, the results of the present study showed a decrease in the expression of GABA Aα1 and GABA B1 in the LGB of neonates of diabetic rats compared with the control group. Numerous studies have shown maternal diabetes may lead to CNS development disorders, which may cause changes in many developmental proceedings such as neurogenesis, migration, differentiation, and cell survival, suggesting that maternal diabetes results in neuropathology through numerous mechanisms (37). Diabetes also declines the neuroprotective function of GABAergic neurons in the cerebral cortex, which causes an increase in neuronal damage in the cerebral cortex during hyperglycemia (13). Diabetes decreases antioxidant levels and concurrently rises the production of free radicals (38). Free radicals such as reactive oxygen species (ROS) can affect ionic homeostasis and neurotransmission. Neurotransmission of GABA is sensitive to ROS (39). The effects of ROS on neurotransmission can happen through numerous mechanisms (40). For example, ROS may interact with neurotransmitter receptors and ion transport channels, causing alterations in receptor activity and ionic homeostasis. The sensitivity of inhibitory neurotransmission, including GABA A , to ROS, is important because changes in GABA neurotransmission can alter the amount of neuronal excitability and it can be one of the reasons for the reduction of GABA following diabetes (38).
GABAergic synaptogenesis is seen in numerous brain areas, and these neurotransmitters have a trophic role through CNS development. In addition, GABA A receptors may provide the stimulus required for growth and differentiation in the early stages of development. GABA exerts its trophic effect during development through depolarizing action (41). In the present study, the reduction of GABA receptors in the LGB of offspring born to diabetic mothers may lead to a reduction of the trophic role of GABA. On the other hand, insulin therapy modulated these changes in the GABA receptor expression in newborns by lowering blood glucose in diabetic mothers.

Effects of maternal diabetes on the expression of mGluR2 receptors in the LGB of neonates
Metabotropic glutamate presynaptic receptors act as autoreceptors throughout the CNS to inhibit glutamate release and reduce glutamate transfer (42). Glutamate has a dual effect on cell survival in the CNS. Low concentrations of glutamate have trophic effects and enhance neuronal survival and synapse formation, while high concentrations are neurotoxic. The neurotoxicity of glutamate is related to calcium signaling (43). Almost every glutamate receptor subtype is involved in excitotoxic cell death. Different receptor subtypes regulate different signal transduction mechanisms that lead to apoptosis (43). Activation of mGluRs changes geniculate relay cell activity by the depolarization of these cells seen during in vitro investigations. Such membrane depolarization controls the activation of a voltage-dependent Ca 2+ channel. It appears the effect of mGluRs on relay cells in the lateral geniculate nucleus is activated only by cortical inputs. (44). Gestational diabetes mellitus (GDM) stimulates microglial activation and chronic inflammatory responses in the brain of the offspring (45). On the other hand, by stimulating microglial activation, mGluR2 expression increased (46). This is probably one of the reasons for the increase in mGluR2 in neonates born to diabetic mothers. An association between diabetes and mGlu receptor expression has been observed in some brain regions, including the hippocampus. Liu, Zh et al. also stated that following the induction of diabetes in the hippocampus, the glutamate content increased, and also the mGluR2/3 subtype receptors are overstimulated. Stimulation of mGluR2/3 suppresses the downstream extracellular signal-regulated kinase (ERK) signaling pathway. ERK modulates downstream pathways that regulate apoptosis. As a result, stimulation of mGluR2-3 decreases ERK expression following diabetes, resulting in the inhibition of apoptotic regulators, and subsequently inducing apoptosis and nerve damage (18). Increased glutamate content is reported to cause neuronal degeneration (47). A study (2004) reported that glutamate and LCCG-I, a specific agonist of the mGluR2 group, reduced the viability of anterior pituitary cells and caused apoptosis in anterior pituitary cells by activating mGluR2 (43). mGluRs can rise cytosolic Ca 2+ stores in neurons. The mGluR-mediated increase in cytosolic Ca 2+ concentrations can activate calcium-sensitive K + channels and calciumdependent non-selective cation channels (48). Ca 2+ is the major second messenger that supports transmitting depolarization status and synaptic activity to a neuron's biochemical machine. Ca 2+ regulation plays a vital role in neurons with extensive and complex signaling pathways (49). In neurons, an increase in local and intracellular Ca 2+ can play an etiological role in neuronal death (50). Also, the present study showed an increase in the expression of mGluR2 in the LGB of neonates of diabetic mothers compared with the control group. This increase in mGluR2 expression may cause an increase in intracellular calcium concentration and neuronal death which can lead to visual symptoms in the future. In the present study, the expression of mGluR2 was decreased in the LGB of rat neonates born to diabetic mothers treated with insulin due to the lowering of blood glucose levels.

Conclusion
According to the results of the present study, it can be concluded that diabetes reduces the expression of GABA Aα1 and GABA B1 receptors but increases the expression of mGluR2 in LGB of male neonates born to diabetic mothers. Due to the important role of these receptors in the visual pathway, changes in the expression of these receptors may cause adverse effects on the visual pathway of neonates of diabetic mothers. Insulin therapy in diabetic mothers can modulate the adverse effects of maternal diabetes in neonates.